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Hyo-Joong Kim's Publications

Under the "RNA World" hypothesis, an early episode of
natural history on Earth used RNA as the only genetically encoded
molecule to catalyze steps in its metabolism catalysis. This, according
to the hypothesis, included RNA catalysts that used RNA cofactors.
However, the RNA World hypothesis places special demands on
prebiotic chemistry, which must now deliver not only four
ribonucleosides, but also must deliver the "functional" portion of these
RNA cofactors. While some (e.g. methionine) present no particular
challenges, nicotinamide ribose is special. Essential to its role in
biological oxidations and reductions, its glycosidic bond that holds a
positively charged heterocycle is especially unstable with respect to
cleavage. Nevertheless, we are able to report here a prebiotic
synthesis of phosphorylated nicotinamide ribose under conditions that
also conveniently lead to the adenosine phosphate components of
this and other RNA cofactors.

According to a current "RNA first" model for the origin of life, RNA
emerged in some form on early Earth to become the first biopolymer
to support Darwinism here. Threose nucleic acid (TNA) and
other polyelectrolytes are also considered as the possible first Darwinian
biopolymer(s). This model is being developed by research
pursuing a "Discontinuous Synthesis Model" (DSM) for the formation
of RNA and/or TNA from precursor molecules that might have
been available on early Earth from prebiotic reactions, with the goal
of making the model less discontinuous. In general, this is done by
examining the reactivity of isolated products from proposed steps
that generate those products, with increasing complexity of the reaction
mixtures in the proposed mineralogical environments. Here,
we report that adenine, diaminopurine, and hypoxanthine nucleoside
phosphates and a noncanonical pyrimidine nucleoside (zebularine)
phosphate can be formed from the direct coupling reaction of
cyclic carbohydrate phosphates with the free nucleobases. The reaction
is stereoselective, giving only the β-anomer of the nucleotides
within detectable limits. For purines, the coupling is also
regioselective, giving the N-9 nucleotide for adenine as a major
product. In the DSM, phosphorylated carbohydrates are presumed
to have been available via reactions explored previously [Krishnamurthy
R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed
39:2281-2285], while nucleobases are presumed to have been available
from hydrogen cyanide and other nitrogenous species formed
in Earth's primitive atmosphere.

One frontier in synthetic biology seeks to move artificially
expanded genetic information systems (AEGIS) into natural living cells and to
arrange the metabolism of those cells to allow them to replicate plasmids built
from these unnatural genetic systems. In addition to requiring polymerases that
replicate AEGIS oligonucleotides, such cells require metabolic pathways that
biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those
polymerases. Such pathways generally require nucleoside and nucleotide kinases
to phosphorylate AEGIS nucleosides and nucleotides on the path to these
triphosphates. Thus, constructing such pathways focuses on engineering natural
nucleoside and nucleotide kinases, which often do not accept the unnatural
AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the
enzyme engineer to follow the kinase reaction, assays that are easily confused by
ATPase and other spurious activities that might arise through "site-directed
damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural
and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring
the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-
(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-
4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro
and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

Reported here is the crystal structure of a heterocycle that implements a donor–donor–acceptor hydrogen-bonding pattern, as found in the Z component [6-amino-5-nitropyridin-2(1H)-one] of an artificially expanded genetic information system (AEGIS). AEGIS is a new form of DNA from synthetic biology that has six replicable nucleotides, rather than the four found in natural DNA. Remarkably, Z crystallizes from water as a 1:1 complex of its neutral and deprotonated forms, and forms a ‘skinny’ pyrimidine–pyrimidine pair in this structure. The pair resembles the known intercalated cytosine pair. The formation of the same pair in two different salts, namely poly[[aqua(µ6-2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ido)sodium]–6-amino-5-nitropyridin-2(1H)-one–water (1/1/1)], denoted Z-Sod, {[Na(C5H4N3O3)(H2O)]·C5H5N3O3·H2O}n, and ammonium 2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ide–6-amino-5-nitropyridin-2(1H)-one–water (1/1/1), denoted Z-Am, NH4+·C5H4N3O3·C5H5-N3O3·H2O, under two different crystallization conditions suggests that the pair is especially stable. Implications of this structure for the use of this heterocycle in artificial DNA are discussed.

Axiomatically, the density of information
stored in DNA, with just four nucleotides (GACT), is
higher than in a binary code, but less than it might be if
synthetic biologists succeed in adding independently
replicating nucleotides to genetic systems. Such addition
could also add additional functional groups, not found in
natural DNA but useful for molecular performance. Here,
we consider two new nucleotides (Z and P, 6-amino-5-
nitro-3-(1'-B-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone
and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo-
[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to
pair via strict Watson?Crick geometry. These were added
to a laboratory in vitro evolution (LIVE) experiment; the
GACTZP library was challenged to deliver molecules that
bind selectively to liver cancer cells, but not to
untransformed liver cells. Unlike in classical in vitro
selection systems, low levels of mutation allow this system
to evolve to create binding molecules not necessarily
present in the original library. Over a dozen binding
species were recovered. The best had Z and/or P in their
sequences. Several had multiple, nearby, and adjacent Zs
and Ps. Only the weaker binders contained no Z or P at all.
This suggests that this system explored much of the
sequence space available to this genetic system and that
GACTZP libraries are richer reservoirs of functionality
than standard libraries.

Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.

As one of its goals, synthetic biology seeks to
increase the number of building blocks in nucleic acids. While
efforts towards this goal are well advanced for DNA, they have
hardly begun for RNA. Herein, we present a crystal structure
for an RNA riboswitch where a stem C:G pair has been
replaced by a pair between two components of an artificially
expanded genetic-information system (AEGIS), Z and P, (6-
amino-5-nitro-2(1H)-pyridone and 2-aminoimidazo[
1,2-a]-1,3,5-triazin-4-(8H)-one). The structure
shows that the Z:P pair does not greatly change
the conformation of the RNAmolecule nor the details
of its interaction with a hypoxanthine ligand. This was
confirmed in solution by in-line probing, which also
measured a 3.7 nm affinity of the riboswitch for
guanine. These data show that the Z:P pair mimics the
natural Watson-Crick geometry in RNA in the first
example of a crystal structure of an RNA molecule
that contains an orthogonal added nucleobase pair.

This year marks the 50th anniversary of a proposal by Alex Rich that RNA, as a single biopolymer acting in two
capacities, might have supported both genetics and catalysis at the origin of life. We review here both published and
previously unreported experimental data that provide new perspectives on this old proposal. The new data include
evidence that, in the presence of borate, small amounts of carbohydrates can fix large amounts of formaldehyde that
are expected in an environment rich in carbon dioxide. Further, we consider other species, including arsenate,
arsenite, phosphite, and germanate, that might replace phosphate as linkers in genetic biopolymers. While linkages
involving these oxyanions are judged to be too unstable to support genetics on Earth, we consider the possibility
that they might do so in colder semi-aqueous environments more exotic than those found on Earth, where cosolvents
such as ammonia might prevent freezing at temperatures well below 273 K. These include the ammonia-water
environments that are possibly present at low temperatures beneath the surface of Titan, Saturn’s largest moon.

6-Aminopyridin-2-ones form Watson-Crick pairs with complementary purine analogues to add a third
nucleobase pair to DNA and RNA, if an electron-withdrawing group at position 5 slows oxidation and epimerization. In previous
work with a nucleoside analogue trivially named dZ, the electron withdrawing unit was a nitro group. Here, we describe an
analogue of dZ (cyano-dZ) having a cyano group instead of a nitro group, including its synthesis, pKa, rates of acid-catalyzed
epimerization, and enzymatic incorporation.

2 '-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2 '-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2 '-deoxycytidine and 2 '-deoxyguanosine pair. (C) 2009 Elsevier Ltd. All rights reserved.

An efficient method for the synthesis of 7-deazaneplanocin A (2) has been accomplished by the condensation of cyclopentenol 3 with 6-chloro-7-deazapurine followed by subsequent functional group manipulations. The synthesized 7-deazaneplanocin A (2) exhibited potent antiviral activity against cowpox and vaccinia viruses without cytotoxicity in HFF cells. (c) 2005 Elsevier Ltd. All rights reserved.

Treatment of (Tp(tBu,Me))YbE(thf) (E=I (1), CH2SiMe3 (2)) with tetramethylimidazol-2-ylidene (ImMe(4)) resulted in very different outcomes depending on the nature of the anionic ligand E. ImMe(4) acts as a simple Lewis base toward 1 resulting in substitution of the thf (tetrahydrofuran) ligand and formation of (Tp(tBu,Me))YbI(ImMe(4)) (3). However, reaction with 2, in addition to displacement of thf, proceeded by metalation of one of the N-CH3 substituents of ImMe(4) and gave (Tp(tBu,Me))Yb(ImMe(4))(CH2N(C(CH3)C(CH3)N(CH3)C) (4), featuring a hydrocarbyl tethered carbene ligand. The X-ray structures of 3 and 4 are reported. (c) 2006 Published by Elsevier B.V.

Synthetic studies of a new family of novel ketonic macrocycles are reported. Exhaustive oxidation of all of the methylenes in odd-numbered [1(n)]orthocyclophanes ([1(n)]OCPs) resulted in the polyoxo derivatives of a cyclopolyorthobenzoyl or polyoxo[1(n)]orthocyclophane constitution. This new class of ketonic crowns is referred to as [1(n)]orthocyclophanepolyones and includes [1(5)] orthocyclophane-pentaone, [1(7)]orthocyclophaneheptaone, and [1(9)]orthocyclophanenonaone. We suggest the generic name ''ketonands'' for these ketonic crowns. Structures of ketonands were confirmed by spectral and X-ray crystallographic analyses.

o-Benzylbenzylic alcohols (o-BBAs), in which the terminal benzyl alcohol is substituted by repeating benzyl chains all in the ortho sense, have been found to have conspicuous regioselectivity in acid-catalysed cycloalkylation, giving rise to various cyclophanes as intramolecular Friedel-Crafts alkylation products. The structure of the cyclisation products was largely dependent upon the size of the benzylic alcohols. Acidic treatment of 2-nuclear o-BBA 6 gave a [1.1]orthocyclophane 7 with a 6-membered ring, whereas 3-nuclear o-BBA 1 afforded [1.1.1]orthocyclophane 2 with a 9-membered ring in preference to a 6-membered-ring product. Higher homologues, such as 4- and 5-nuclear o-BBAs, gave rise to [1.(4)](1,2)(1,2)(1,2)(1,3)cyclophanes 14 and 25 with a 13-membered ring unit, respectively. Cyclophanes with a larger-than-1 3-membered ring have never been isolated as cycloalkylation products of o- BBA. Generalisations have been made about the priority of formation of cycles in the cycloalkylation of o-BBA in acid, to give a cycloalkylation rule, which involves the priority order of 13-membered ring > 9-membered ring > 6-membered ring. The regioselectivity was consistent with the acid-catalysed cycloalkylation of alpha,omega-benzylbenzylic diols, which yielded common-nuclear biscyclophanes. The sizes and structures of the biscyclophane products are also dependent upon the sizes and structures of the terminal benzylic diols.